The field of this invention is erythromycin production. More particularly, the present invention pertains to a method of improving the strain used for the production of erythromycin through the disruption of the melA gene.
Actinomycete fermentations are the source of many medically important pharmaceuticals, particularly antibiotics. The commercial production of these compounds is made more economical through genetic alterations in the producing organism, referred to as strain improvements, that are traditionally introduced through a random mutation and screening process (Queener, S. W. and D. H. Lively 1986. Screening and selection for strain improvement, p. 155-169. In Manual of Industrial Microbiology and Biotechnology. Eds. A. L. Demain and N. A. Solomon. American Society for Microbiology, Washington. 1986). The traditional process is tedious and time consuming, but is technically simple to perform. Its major drawback is that it is empirical; and during the 50 years that it has been practiced by industry, very little has been learned concerning the genetics of strain improvement.
More recently molecular genetic technology has been developed that allows for the introduction of xe2x80x9ctargetedxe2x80x9d genetic alterations of industrially important strains. In particular, the erythromycin producing strain, Sac. erythraea, has a well developed system for integrative transformation, targeted gene replacement and disruption (Weber, J. M. and R. Losick, 1988, Gene 68, 173-180; Weber, J. M., J. O. Leung, G. T. Maine, R H. B. Potenz, T. J. Paulus and J. P. DeWitt, 1990, J. Bacteriol. 172, 2372-2383). This approach, though technically more difficult to perform, provides yield improvement results plus insight into the metabolic and genetic events that lead to strain improvement.
Although molecular genetic technology has been used in Sac. erythraea for the development of novel macrolide structures (Cortes, J., K. E. Wiesmann, G. A. Roberts, M. J. Brown, J. Staunton, and P. F. Leadlay, 1995, Science 268:1487-1489.; Donadio, S., J. B. McAlpine, P. A. Sheldon, M. A. Jackson, L. Katz, 1993, PNAS USA 90:7119-7123), it has not yet been applied to the area of erythromycin strain improvement.
Current strain improvement technology consists of an empirical and labor intensive process of introducing randomly produced mutations followed by large-scale brute-force screening for better strains. Targeted gene disruption is a way to rationally modify a strain of Saccharopolyspora to overproduce erythromycin. Currently there are no other genes described whose inactivation will lead specifically and reproducibly to an improved erythromycin-producing strain. Erythromycin is a bulk pharmaceutical produced in the thousands of metric tons per year and the market for this bulk compound is approximately 600 million dollars per year. Any improvement in the production process that would lead to substantial increases in production would have significant economic implications.
The method of the invention, herein described, includes the genetic modification of an erythromycin-producing microorganism through the targeted disruption of the melA gene with plasmid pFL1046 so that the microorganism is transformed into a more efficient and more robust producer of erythromycin under conditions where oxygen is a limiting nutrient. Plasmid pFL1046 is a derivative of plasmid pFL14 which was isolated from a library of Sac. erythraea DNA fragments found during a visual blue-pigment screening procedure in S. lividans. The DNA sequence of a subclone of pFL14, pFL1040, is shown in FIG. 1 (SEQ ID NO:1) showing the coding sequence of the melA gene (SEQ ID NO:2) from Sac. erythraea. The alignment of the deduced amino acid sequence of the melA gene (SEQ ID NO:3) from Sac. erythraea is compared to the sequence of melA genes from other organisms (FIG. 3 SEQ ID NOS:6-11). A very high degree of homology is seen to these other melA genes which further supports the fact that this gene is in fact involved in pigment biosynthesis in Sac. erythraea. 
According to one aspect of the method of the invention, transformation of an erythromycin-producing microorganism into a more robust producer is accomplished by integrating, via homologous recombination, a plasmid constructed from a parent vector, pFL8 and a DNA fragment from the Sac. erythraea chromosome which is internal to the coding sequence of the 4-hydroxyphenylpyrivic acid dioxygenase (melA) gene. Integrative transformation of this plasmid into the Sac. erythraea chromosome disrupts the normal function of the melA gene which consequently blocks the production of pyomelanin pigment and slows the growth of the organism. This integrative plasmid is constructed to be capable of being stably maintained in the microorganism (i.e., of being passed faithfully in its active form from one generation to the next).
A microorganism embodying the present invention is a novel strain of Sac. erythraea with lower oxygen requirements for the production of erythromycin in an aqueous medium containing assimilable sources of nitrogen and carbon. The blockage of metabolic flow of oxygen into pigment biosynthesis and tyrosine metabolism reduces the strains requirement for oxygen, and indirectly slows the growth of the strain, but does not negatively affect erythromycin biosynthesis.